Molecular arrays are widely used and increasingly important tools for rapid hybridization analysis of sample solutions against hundreds or thousands of precisely ordered and positioned features containing different types of molecules within the molecular arrays. Molecular arrays are normally prepared by synthesizing or attaching a large number of molecular species to a chemically prepared substrate such as silicone, glass, or plastic. Each feature, or element, within the molecular array is defined to be a small, regularly-shaped region of the surface of the substrate. The features are arranged in a regular pattern. Each feature within the molecular array may contain a different molecular species, and the molecular species within a given feature may differ from the molecular species within the remaining features of the molecular array. In a hybridization experiment, a sample solution containing radioactively, fluorescently, or chemoluminescently labeled molecules is applied to the surface of the molecular array. Certain of the labeled molecules in the sample solution may specifically bind to, or hybridize with, one or more of the different molecular species that together comprise the molecular array. Following hybridization, the sample solution is removed by washing the surface of the molecular array with a buffer solution, and the molecular array is then analyzed by radiometric or optical methods to determine to which specific features of the molecular array the labeled molecules are bound. Thus, in a single experiment, a solution of labeled molecules can be screened for binding to hundreds or thousands of different molecular species that together comprise the molecular array. Molecular arrays commonly contain oligonucleotides or complementary deoxyribonucleic acid ("cDNA") molecules to which labeled deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") molecules bind via sequence-specific hybridization.
Molecular arrays are generally rather large, commonly having surface areas of between 4 and 16 cm.sup.2. The volumes of solutions of labeled molecules applied to the surfaces of molecular arrays may be quite small, on the order of between 50 and 100 .mu.L. Thus, when an applied solution is evenly distributed across the surface of a molecular array, the applied solution may have a thickness of anywhere between thirty and several hundred microns.
There are a number of problems associated with manipulating small volumes of sample solution during the course of a hybridization experiment. First, the sample solution must be evenly distributed across the surface of the molecular array. Second, in many types of hybridization experiments, the sample solution, once distributed across the surface of the molecular array, needs to be enclosed within a sealed container in order to prevent evaporation of the sample solution during the time required for hybridization to occur. Third, once hybridization has completed, any remaining unbound labeled molecules must be rinsed from the surface of the molecular array. Finally, in many hybridization experiments, the surface of the molecular array must remain hydrated by a buffer solution until binding of labeled molecules to the surface of the molecular array is detected by radiometric or optical methods. It is desirable for the entire process to be automated to as great an extent as possible in order to increase the accuracy and throughput of hyridization screening.
Currently, experimenters may use a manual approach to solve these problems. A silicone gel or other such non-reactive, water impermeable substance may be placed along the edges of the molecular array, sample solution placed onto the center of the molecular array, and a glass cover slip placed on top of the sample solution in order to press the sample solution down towards the surface of the molecular array and distribute it across the surface of the molecular array. The glass cover slip makes contact with the silicone gel at the edges of the molecular array to form a seal enclosing the sample solution between the surface of the molecular array, the glass cover slip, and the silicone gel. However, this method is tedious and time consuming, especially in view of the fact that the glass cover slip must be removed in order to rinse the surface of the molecular array prior to radiometric or optical analysis. Another approach is to embed the molecular array within the surface of a plastic housing with a continuous side wall that forms an open well, or container, with the molecular array at the bottom of the well. After the sample solution is applied to the molecular array, the plastic housing is covered with a lid to form a seal enclosing a relatively small volume above the surface of the molecular array, and the housing and molecular array is then spun in a centrifuge to apply centrifugal force to the sample solution in order to spread the sample solution evenly across the surface of the molecular array. Once distributed across the surface of the molecular array, capillary action holds the sample solution to the surface of the molecular array during the time required for hybridization. Following hybridization, the lid is removed from the plastic housing in order to rinse away any remaining unbound, unlabeled molecules, and a replacement buffer solution is added to the container, followed by replacement of the lid.
While more amenable to automation, the second technique may limit the accuracy and reliability of optical analysis of fluorescently labeled, bound sample molecules. In common optical analysis techniques, a pinpoint laser beam scans the surface of the molecular array in order to excite fluorophores bound to sample molecules hybridized with molecular array molecules. The laser beam passes through the molecular array substrate in order to reach the bound molecules, continues into the buffer solution distributed across the surface of the molecular array, and is finally absorbed by the lid enclosing the well or container in a seal. Because the sample volumes are small, the lid is positioned quite close to the inner surface of the molecular array, on an order of 3 to 5 thousandths of an inch. The laser beam, under these conditions, may effectively excite naturally occurring fluorophores within the lid as well as fluorophores bound to sample molecules. When fluorescence is subsequently measured by optically focused measuring devices, fluorescence from the naturally occurring fluorophores within the lid may add significant background to the fluorescence emanating from the fluorophores bound to sample molecules, decreasing the signal-to-noise ratio of the measured fluorescent emissions from the sample molecules. To decrease the background fluorescence emanating from the lid, the lid may be manufactured from materials chosen to have very low concentrations of natural fluorophores. Glass is one such substance, but the manufacture and manipulation of glass lids may be expensive and prone to mechanical damage. Another approach is to add light-absorbing filler, such as carbon black, to plastics in order to absorb fluorescent emissions internally within the lid. However, this approach has failed to sufficiently reduce the contribution to background fluorescence from naturally occurring fluorophores in the lid. Thus, manufacturers of molecular arrays and experimenters using molecular arrays for hybridization screening have recognized the need for a straightforward technique and apparatus, amenable to automation, for manipulating small volumes of sample solutions tightly sealed within close proximity to the surfaces of molecular arrays and for optically measuring the fluorescence of fluorescently labeled sample molecules bound to the surface of molecular arrays.